ENT MERFISH Report

1. Overview

1.1 Sample Information

A brief sample information is generated from the submission table for the following analysis.

Sample Index and Basic Information
Expt Sample Index Case_Number Sex Age HPV Sample_Region Genotype Group Region DataPath Clinic.comment
5 2 TT29258 TT29258 3 M 47 Positive Right tongue cancer TT Tumor region_0 Y:_Imaging_data_2\202412061704_20241206ENT2925859ImmunOncoh500x03_VMSC00101 right tongue cancer, involving the right hemitongue and base of tongue, cT4N2bM0
6 2 NT29258 NT29258 3 M 47 Positive Right tongue cancer NT Normal region_1 Y:_Imaging_data_2\202412061704_20241206ENT2925859ImmunOncoh500x03_VMSC00101 right tongue cancer, involving the right hemitongue and base of tongue, cT4N2bM0

1.2 MERSCOPE Data Quality Summary

The summaries present the data quality assessment automatically generated by MERSCOPE for each experiment. We mainly focus on the transcripts level for each sample. So we’re looking for high density in transcripts, based on the transcripts count per field of view (FOV), transcript density in FOV, and frequency of transcripts detected.

Generally, log10 transcript count > 4.0 in most area can be considered as a good quality standard for human tissue.

Need to note that the low accuracy in DAPI cell boundary is not a concern, as a self-designed cell segmentation processing will take over this task.

1.2.1 TT29258(Tumor)

1.2.2 NT29258(Normal)

2. Data Processing & Analysis

2.1 Cell Segmentation & Filtering

Based on the spatial information and images obtained from MERFISH, we developed a machine learning model using the Cellpose algorithm to distinguish individual cells via MERFISH DAPI images.

To ensure the data quality and accuracy of cells, we have defined the minimum and maximum values for cell volume and gene count per cell. The cell volume should be between [100, 2000], and the gene count per cell > 25.

After that, we successfully obtained high-quality and accurate cell and gene data. We are now ready to perform statistical analysis.

For analysis platform, all of the analysis and visualization are performed using Scanpy, a commonly used Python package toolkit for analyzing single-cell gene expression data.

2.1.1 Transcript Count Violin

The transcript level is a basic aspect of data quality control. After filtering, we have ensured that the minimum transcript count per cell > 25.

Transcript Count Violin After Filtering

Transcript Count Violin After Filtering

2.2 Dimension Reduction

We conducted Leiden UMAP clustering, a non-linear, manifold-aware dimension reduction algorithm, on our dataset to subset the cells into clusters. The following annotations are based on the umap clustering.

Umap of cells and colored by batch

Umap of cells and colored by batch

3.Cell Annotation

To annotate individual cell types, we found the marker genes from database CellMarker.

It is good to note that, so far what we have is a preliminary annotation, and we can potentially achieve further resolution on some cell types. For instance, T cells can be subdivided into regulatory T cells, helper T cells, natural killer T cells, and so on. However, the current sample size is insufficient for these detailed classifications. Once we have enough samples, further annotation should be feasible.

3.1 Markers for type annotation

3.2 Cell Type Umap

3.3 Cell Type Spatial Map

With the annotations, we can map cell types back to their physical positions and create a spatial map for each sample.

3.4 Cell Type Qualification Analysis

Cell Type Count
cell_type_1 NT29258 TT29258 Total
B cell 6 4878 4884
Endothelial cell 2620 2645 5265
Fibroblast 6022 8327 14349
Macrophage 657 1456 2113
Mast cell 75 139 214
Myeloid cell 2745 1880 4625
T cell 156 902 1058
Tumor 9 5556 5565
Total 12290 25783 38073

3.5 Cell Type Proportion Analysis

Based on the quantification of individual cell types, we are capable to compare the differences in cell numbers between various samples and genotypes. The proportion of each cell type is calculated by dividing the count of that specific cell type by the total cell count. The results are then visualized in a bar plot.

4. Gene differentiation (Tumor sample v.s. Normal sample)

Here, we use Wilcoxon rank-sum test to compute gene differential expression (DE). P value are adjusted using Benjamini–Hochberg procedure. The statistical significance was cut-off by log2(Fold Change) > 1 or log2(Fold Change) < -1 and adjusted p_value < 0.05.

The result are visualized via Volcano plot: a type of scatterplot that shows statistical significance (P value) versus magnitude of change (fold change).

The comparison is between Tumor and Normal samples. A positive fold change means higher expression in the tumor group.

4.1 all

X p_val avg_log2FC pct.1 pct.2 p_val_adj
LYZ 0.00e+00 8.553653 0.521 0.014 0.0000000
SOCS3 0.00e+00 -1.161272 0.324 0.644 0.0000000
XBP1 0.00e+00 4.361607 0.393 0.074 0.0000000
MYC 0.00e+00 -1.309076 0.247 0.528 0.0000000
MZB1 0.00e+00 7.467046 0.211 0.007 0.0000000
POU2AF1 0.00e+00 6.571428 0.183 0.008 0.0000000
DES 0.00e+00 -1.267858 0.106 0.273 0.0000000
NRP1 0.00e+00 -1.504230 0.093 0.258 0.0000000
PDK4 0.00e+00 -2.616844 0.084 0.249 0.0000000
SOX9 0.00e+00 3.188959 0.179 0.038 0.0000000
DERL3 0.00e+00 5.817783 0.147 0.009 0.0000000
FCRL5 0.00e+00 7.223381 0.116 0.002 0.0000000
CD79A 0.00e+00 5.387722 0.110 0.006 0.0000000
NCAM1 0.00e+00 -1.474317 0.063 0.177 0.0000000
IRF4 0.00e+00 4.743396 0.106 0.009 0.0000000
THBD 0.00e+00 -1.096753 0.091 0.219 0.0000000
EPHB3 0.00e+00 3.987145 0.109 0.013 0.0000000
ICAM3 0.00e+00 3.332113 0.122 0.020 0.0000000
ITGA5 0.00e+00 -1.257460 0.076 0.191 0.0000000
PIK3CG 0.00e+00 3.895128 0.116 0.019 0.0000000
FN1 0.00e+00 -1.068698 0.152 0.291 0.0000000
CAV1 0.00e+00 -1.580691 0.042 0.130 0.0000000
LRP5 0.00e+00 1.938492 0.258 0.131 0.0000000
SH2D1B 0.00e+00 -3.214215 0.008 0.062 0.0000000
TNFRSF13C 0.00e+00 4.149630 0.089 0.011 0.0000000
AKT3 0.00e+00 -1.280997 0.041 0.117 0.0000000
CD27 0.00e+00 4.387923 0.069 0.005 0.0000000
TGFBR3 0.00e+00 -1.112405 0.065 0.149 0.0000000
VEGFA 0.00e+00 -1.234113 0.321 0.427 0.0000000
SOX2 0.00e+00 2.431169 0.088 0.019 0.0000000
IL6 0.00e+00 -1.896159 0.029 0.087 0.0000000
CDH5 0.00e+00 -1.268976 0.047 0.118 0.0000000
NEDD4 0.00e+00 -1.517832 0.028 0.087 0.0000000
CXCR4 0.00e+00 2.345947 0.103 0.030 0.0000000
CDH1 0.00e+00 3.036970 0.060 0.007 0.0000000
IFNGR1 0.00e+00 -1.922751 0.010 0.048 0.0000000
GATA3 0.00e+00 2.832913 0.062 0.010 0.0000000
CCL28 0.00e+00 4.199291 0.051 0.005 0.0000000
PREX2 0.00e+00 -1.474691 0.024 0.074 0.0000000
ZEB1 0.00e+00 -1.206880 0.036 0.090 0.0000000
TEAD4 0.00e+00 -1.044913 0.040 0.097 0.0000000
CSF3 0.00e+00 -1.459452 0.022 0.065 0.0000000
ERBB3 0.00e+00 2.081145 0.060 0.013 0.0000000
LDHA 0.00e+00 -1.212581 0.030 0.077 0.0000000
CCL2 0.00e+00 -1.787686 0.018 0.055 0.0000000
ERBB2 0.00e+00 1.332449 0.168 0.102 0.0000000
IRF3 0.00e+00 1.162201 0.167 0.100 0.0000000
KLF2 0.00e+00 1.210445 0.248 0.183 0.0000000
EPHA4 0.00e+00 -1.426404 0.014 0.044 0.0000000
TNFRSF18 0.00e+00 3.227918 0.038 0.007 0.0000000
TNFRSF13B 0.00e+00 3.581888 0.032 0.004 0.0000000
PDK1 0.00e+00 1.745148 0.090 0.044 0.0000000
COL6A3 0.00e+00 -1.531173 0.008 0.030 0.0000000
TP63 0.00e+00 -1.073500 0.024 0.056 0.0000000
TGFB1 0.00e+00 1.121922 0.147 0.094 0.0000000
WNT3A 0.00e+00 -1.052591 0.025 0.056 0.0000000
TNFRSF17 0.00e+00 2.232042 0.036 0.009 0.0000000
ITGA1 0.00e+00 -1.703597 0.006 0.024 0.0000000
CDKN1B 0.00e+00 1.121936 0.140 0.089 0.0000000
ITGB2 0.00e+00 1.294243 0.077 0.037 0.0000000
PPARGC1A 0.00e+00 -1.219482 0.014 0.037 0.0000000
MET 0.00e+00 -1.076807 0.020 0.047 0.0000000
ANGPT2 0.00e+00 -1.226841 0.012 0.034 0.0000000
TGFBR1 0.00e+00 -1.039379 0.015 0.038 0.0000000
TEK 0.00e+00 -1.216011 0.011 0.030 0.0000000
TWIST1 0.00e+00 -1.124445 0.020 0.044 0.0000000
S100A9 0.00e+00 -1.132742 0.026 0.052 0.0000000
RORC 0.00e+00 1.055291 0.167 0.122 0.0000000
SELPLG 0.00e+00 2.039868 0.030 0.009 0.0000000
FGF2 0.00e+00 -1.015094 0.014 0.033 0.0000000
CLCA1 0.00e+00 -1.124602 0.012 0.028 0.0000000
CD19 0.00e+00 2.589916 0.019 0.005 0.0000000
HGF 0.00e+00 -1.137755 0.007 0.020 0.0000000
CXCL5 0.00e+00 2.362657 0.024 0.008 0.0000000
TBK1 0.00e+00 -1.037875 0.008 0.020 0.0000000
CTSW 0.00e+00 1.689443 0.036 0.017 0.0000000
BLK 0.00e+00 1.871567 0.030 0.014 0.0000000
CCL5 0.00e+00 1.864179 0.025 0.011 0.0000000
RB1 0.00e+00 -1.317659 0.004 0.011 0.0000000
RET 0.00e+00 -1.080512 0.006 0.015 0.0000000
PDCD1 0.00e+00 1.700711 0.016 0.006 0.0000000
CD3E 0.00e+00 1.700278 0.021 0.009 0.0000000
CCL4 0.00e+00 2.208467 0.018 0.007 0.0000000
KITLG 0.00e+00 -1.405904 0.003 0.011 0.0000000
PLOD2 0.00e+00 1.607495 0.024 0.011 0.0000000
CD22 0.00e+00 1.620492 0.026 0.013 0.0000000
CD79B 0.00e+00 1.074239 0.046 0.029 0.0000000
CA7 0.00e+00 -1.089302 0.006 0.014 0.0000000
MYBL2 0.00e+00 1.255196 0.044 0.028 0.0000000
BAX 0.00e+00 1.015571 0.051 0.034 0.0000000
TRAC 0.00e+00 1.676150 0.016 0.007 0.0000000
ZAP70 0.00e+00 1.067668 0.048 0.032 0.0000000
TBX21 0.00e+00 1.129233 0.035 0.021 0.0000000
TGM2 0.00e+00 -1.134958 0.004 0.010 0.0000000
CEACAM1 0.00e+00 1.975467 0.010 0.003 0.0000000
LAMB3 0.00e+00 1.200616 0.030 0.018 0.0000000
PDGFC 0.00e+00 1.114371 0.030 0.018 0.0000000
SERPINA1 0.00e+00 1.970176 0.011 0.004 0.0000000
CCR10 0.00e+00 1.058810 0.035 0.022 0.0000000
CD5 0.00e+00 1.185667 0.034 0.022 0.0000000
CCR7 0.00e+00 1.860615 0.020 0.011 0.0000000
TNFRSF4 0.00e+00 1.271639 0.019 0.011 0.0000001
TNFRSF9 0.00e+00 1.093588 0.022 0.013 0.0000001
IL2RB 0.00e+00 1.511024 0.020 0.011 0.0000003
XCR1 1.40e-06 1.210747 0.011 0.006 0.0006773
TRBC1 6.00e-06 1.377604 0.013 0.008 0.0029777
ITGAX 7.30e-06 1.195137 0.017 0.011 0.0036746
CCR6 2.87e-05 1.072633 0.012 0.008 0.0143375

4.2 Endothelial cell

X p_val avg_log2FC pct.1 pct.2 p_val_adj
LYZ 0.00e+00 5.823706 0.358 0.018 0.0000000
CAV1 0.00e+00 -1.094243 0.173 0.358 0.0000000
ITGB1 0.00e+00 -1.180874 0.095 0.235 0.0000000
EPHA4 0.00e+00 -1.066876 0.065 0.150 0.0000000
IFNGR1 0.00e+00 -2.075553 0.010 0.060 0.0000000
TEK 0.00e+00 -1.273894 0.040 0.106 0.0000000
INSR 0.00e+00 1.090267 0.218 0.132 0.0000000
S100A9 0.00e+00 -2.541226 0.010 0.048 0.0000000
CCL2 0.00e+00 -1.560417 0.029 0.078 0.0000000
ITGA1 0.00e+00 -1.333629 0.020 0.064 0.0000000
IL6 0.00e+00 -1.095213 0.066 0.125 0.0000000
CXCL2 0.00e+00 -1.174474 0.029 0.068 0.0000000
LDHA 0.00e+00 -1.106462 0.022 0.052 0.0000032
TBK1 3.00e-07 -1.386603 0.005 0.020 0.0001368
KRAS 1.30e-06 -1.398507 0.008 0.026 0.0006329
TBX3 3.10e-06 -1.121951 0.016 0.036 0.0015674
IFNAR1 4.90e-06 -3.273683 0.001 0.010 0.0024308
MZB1 1.27e-05 2.331646 0.026 0.010 0.0063748
PCNA 1.97e-05 -2.794090 0.002 0.011 0.0098585
XBP1 2.18e-05 1.246779 0.084 0.056 0.0108810
FLI1 4.03e-05 -1.429985 0.004 0.015 0.0201280
POU2AF1 4.55e-05 2.506649 0.016 0.005 0.0227664

4.3 Fibroblast

X p_val avg_log2FC pct.1 pct.2 p_val_adj
LYZ 0.00e+00 6.419445 0.330 0.010 0.0000000
SOCS3 0.00e+00 -1.011173 0.394 0.629 0.0000000
NRP1 0.00e+00 -1.102515 0.175 0.353 0.0000000
TGFBR3 0.00e+00 -1.140151 0.102 0.230 0.0000000
S100A9 0.00e+00 -4.078093 0.005 0.063 0.0000000
ITGA5 0.00e+00 -1.005786 0.087 0.175 0.0000000
MZB1 0.00e+00 4.336611 0.050 0.005 0.0000000
COL6A3 0.00e+00 -1.869007 0.014 0.053 0.0000000
IL6 0.00e+00 -1.343519 0.055 0.114 0.0000000
ZEB1 0.00e+00 -1.128739 0.046 0.102 0.0000000
XBP1 0.00e+00 1.740505 0.109 0.051 0.0000000
CDK2 0.00e+00 -1.015565 0.046 0.098 0.0000000
PREX2 0.00e+00 -1.123112 0.031 0.074 0.0000000
WNT5A 0.00e+00 -1.727531 0.017 0.050 0.0000000
IFNGR1 0.00e+00 -1.476772 0.013 0.042 0.0000000
MAFB 0.00e+00 -1.059271 0.050 0.095 0.0000000
SNAI2 0.00e+00 -1.045211 0.036 0.075 0.0000000
MSH6 0.00e+00 -1.092429 0.022 0.053 0.0000000
TGFBR1 0.00e+00 -1.119738 0.016 0.042 0.0000000
RET 0.00e+00 -2.087603 0.006 0.024 0.0000000
MSH3 0.00e+00 -1.046197 0.020 0.044 0.0000000
TWIST1 0.00e+00 -1.026474 0.031 0.060 0.0000000
CD79A 0.00e+00 3.089908 0.019 0.004 0.0000000
CCL2 0.00e+00 -1.035696 0.027 0.051 0.0000000
POU2AF1 0.00e+00 2.026522 0.028 0.010 0.0000000
STAT4 0.00e+00 -1.160355 0.012 0.028 0.0000000
MET 0.00e+00 -1.254647 0.013 0.029 0.0000000
TBK1 0.00e+00 -1.402651 0.006 0.018 0.0000001
KITLG 3.00e-07 -1.166638 0.006 0.014 0.0001382
C1QC 2.50e-06 1.372830 0.014 0.006 0.0012648
CIITA 4.10e-06 1.241280 0.034 0.021 0.0020568
SERPINE1 1.47e-05 1.003024 0.113 0.094 0.0073496
EPHA4 1.96e-05 -1.016471 0.006 0.013 0.0097784

4.4 Macrophage

X p_val avg_log2FC pct.1 pct.2 p_val_adj
LYZ 0.00e+00 4.666015 0.359 0.043 0.0000000
MRC1 0.00e+00 -1.143962 0.408 0.616 0.0000000
MYC 0.00e+00 -1.490831 0.164 0.355 0.0000000
CCR1 0.00e+00 -1.583498 0.055 0.175 0.0000000
CCL2 0.00e+00 -2.127889 0.041 0.132 0.0000000
S100A9 0.00e+00 -2.422379 0.038 0.120 0.0000000
MZB1 0.00e+00 5.650988 0.087 0.006 0.0000000
CCL8 0.00e+00 -1.951505 0.027 0.085 0.0000008
CCL4 0.00e+00 1.762221 0.124 0.041 0.0000012
ITGAX 0.00e+00 1.786399 0.192 0.096 0.0000012
TGFBR1 0.00e+00 -1.697928 0.020 0.068 0.0000110
CD209 0.00e+00 -1.242842 0.073 0.149 0.0000155
POU2AF1 3.00e-07 3.763569 0.052 0.006 0.0001674
IFNGR1 4.10e-06 -2.269987 0.012 0.044 0.0020712
CCL3 1.82e-05 1.241680 0.130 0.068 0.0091155
EZH2 2.40e-05 -2.248266 0.005 0.026 0.0119791
CAV1 2.52e-05 -2.009138 0.012 0.040 0.0126185
CD27 3.94e-05 3.616486 0.029 0.002 0.0196895
MMRN1 5.65e-05 4.241767 0.031 0.003 0.0282728
IL12RB2 6.08e-05 -2.395933 0.005 0.024 0.0303982

4.5 Mast cell

X p_val avg_log2FC pct.1 pct.2 p_val_adj
KIT 0.00e+00 -1.598224 0.691 0.973 0.0000000
NFKB2 0.00e+00 -1.536690 0.482 0.800 0.0000002
JUN 0.00e+00 2.167337 0.640 0.293 0.0000105
CSF1 1.00e-07 2.004793 0.719 0.427 0.0000308
FOS 1.00e-07 1.342368 0.820 0.600 0.0000353
SOCS3 2.00e-06 -1.669082 0.173 0.467 0.0010165
CTSG 1.38e-05 -1.150132 0.561 0.813 0.0068827
CTNNB1 1.75e-05 -1.974737 0.101 0.333 0.0087389
CD274 2.43e-05 -1.679176 0.101 0.347 0.0121748
CDK4 6.58e-05 -5.435495 0.007 0.133 0.0328979

4.6 Myeloid cell

X p_val avg_log2FC pct.1 pct.2 p_val_adj
PDK4 0.00e+00 -1.816208 0.432 0.822 0.0000000
LYZ 0.00e+00 5.927489 0.305 0.014 0.0000000
CDKN1A 0.00e+00 1.980209 0.646 0.264 0.0000000
VEGFA 0.00e+00 -1.016840 0.691 0.921 0.0000000
DUSP1 0.00e+00 1.434790 0.665 0.454 0.0000000
SH2D1B 0.00e+00 -2.830498 0.044 0.262 0.0000000
BCL2L1 0.00e+00 1.597147 0.416 0.198 0.0000000
CSF3 0.00e+00 -1.638053 0.053 0.172 0.0000000
ELN 0.00e+00 1.556845 0.170 0.076 0.0000000
NFKB2 0.00e+00 1.076388 0.279 0.169 0.0000000
TGFB1 0.00e+00 2.412329 0.071 0.020 0.0000000
SNAI2 0.00e+00 2.785764 0.053 0.011 0.0000000
COL1A1 0.00e+00 1.337087 0.129 0.059 0.0000000
FZD7 0.00e+00 -1.437161 0.045 0.115 0.0000000
BMP1 0.00e+00 1.309168 0.157 0.083 0.0000000
ATF3 0.00e+00 1.657287 0.108 0.046 0.0000000
LDHA 0.00e+00 -1.096763 0.055 0.123 0.0000000
DERL3 0.00e+00 2.347748 0.052 0.016 0.0000000
PLVAP 0.00e+00 1.228717 0.089 0.042 0.0000000
LRP1 0.00e+00 1.029352 0.140 0.081 0.0000000
TGFBR2 0.00e+00 1.055792 0.122 0.067 0.0000000
PDGFRB 0.00e+00 1.398942 0.070 0.030 0.0000001
PDPN 0.00e+00 1.380385 0.080 0.039 0.0000003
SERPINE1 0.00e+00 1.309020 0.040 0.014 0.0000047
VSIR 0.00e+00 -1.630982 0.013 0.042 0.0000098
HLA-DRA 0.00e+00 1.677830 0.046 0.018 0.0000128
ITGAM 0.00e+00 1.170689 0.084 0.044 0.0000130
PLA2G2A 0.00e+00 1.227550 0.064 0.030 0.0000173
COL4A1 0.00e+00 1.337863 0.065 0.032 0.0000193
BCL2 2.00e-07 -1.185583 0.039 0.077 0.0000759
HLA-B 2.00e-07 1.461630 0.053 0.024 0.0000920
NRP1 2.00e-07 -1.616306 0.019 0.047 0.0001074
ADAMTS4 3.00e-07 1.772789 0.028 0.008 0.0001654
IFNGR1 6.00e-07 -1.024887 0.022 0.051 0.0003024
SFRP2 4.90e-06 1.083273 0.047 0.023 0.0024408
CLDN5 5.00e-06 1.222015 0.071 0.041 0.0024951
RB1 6.00e-06 -2.125089 0.004 0.019 0.0030059
CHEK2 6.70e-06 2.801931 0.018 0.004 0.0033741
IL23A 1.40e-05 -1.277117 0.016 0.039 0.0070196
STING1 1.68e-05 1.378475 0.037 0.017 0.0084026
MMP2 1.74e-05 1.851327 0.021 0.007 0.0086764
SOX9 2.60e-05 2.179544 0.024 0.009 0.0129791

4.7 T cell

X p_val avg_log2FC pct.1 pct.2 p_val_adj
LMNA 0.00e+00 -2.445791 0.154 0.615 0.0000000
LYZ 0.00e+00 5.603927 0.385 0.026 0.0000000
S100A9 0.00e+00 -2.543922 0.022 0.147 0.0000000
MZB1 1.00e-07 5.816204 0.180 0.013 0.0000442
TP63 1.10e-05 -2.798094 0.003 0.038 0.0054981
ITGA4 1.15e-05 -2.024359 0.025 0.096 0.0057748
AKT3 1.41e-05 -1.703896 0.039 0.122 0.0070505
POU2AF1 2.32e-05 5.165136 0.126 0.013 0.0115835
KLF2 3.22e-05 1.710957 0.258 0.109 0.0160790
CTNNB1 3.50e-05 -1.274266 0.108 0.224 0.0175073
HLA-DPB1 3.73e-05 1.368051 0.259 0.109 0.0186749
BST2 5.13e-05 2.082583 0.187 0.058 0.0256729
CIITA 5.36e-05 2.278486 0.151 0.032 0.0268148
ICOS 6.34e-05 -2.474700 0.009 0.051 0.0317023

5. Supplements

For original pictures and more details, please use the google drive link: ENT MERFISH Supplement